Hubrecht Institute Centennial - From embryos to stem cells.

During the last 100 years, the Hubrecht Institute evolved from a small laboratory aimed at providing research material to the scientific community to a modern, fully equipped research institute with state-of-the-art infrastructure, performing research at the highest standard. The past 100 years have been eventful for the Hubrecht Institute with many glorious moments, but also threats to be shut down on several occasions. Here, we will briefly review the rich history of the Hubrecht Institute.

Efficient evaluation of Sommerfeld integrals for the optical simulation of many scattering particles in planarly layered media.

A strategy for the efficient numerical evaluation of Sommerfeld integrals in the context of electromagnetic scattering at particles embedded in a plane parallel layer system is presented. The scheme relies on a lookup-table approach in combination with an asymptotic approximation of the Bessel function in order to enable the use of fast Fourier transformation. Accuracy of the algorithm is enhanced by means of singularity extraction and a novel technique to treat the integrand at small arguments. For short particle distances, this method is accomplished by a slower but more robust direct integration along a deflected contour. As an example, we investigate enhanced light extraction from an organic light-emitting diode by optical scattering particles. The calculations are discussed with respect to accuracy and computing time. By means of the present strategy, an accurate evaluation of the scattered field for several thousand wavelength scale particles can be achieved within a few hours on a conventional workstation computer.

Sensitivity improvement in fluorescence-based particle detection.

Microfluidic flow cytometers are highly interesting candidates for biomedical point-of-care applications. However, the sensitivity, reliability, and throughput of these systems must be improved to provide the full functionality of established flow cytometric systems. One proposed method to improve fluorescence detection systems is to use spatial modulation techniques. We derive the noise-related statistics and calculate the coefficient of variation for a detection system with and without spatial modulation. We measure the noise properties of a nonmodulated microfluidic fluorescence particle detection system and analyze the possible performance gains using spatial modulation.

Comorbidities in overweight children and adolescents: do we treat them effectively?

OBJECTIVE: The aim was to analyze the effectiveness of treatment concerning obesity-associated comorbidities in clinical practice. METHODS: A total of 11,681 overweight children with ≥ 6-month follow-up treated at 175 centers specialized in pediatric obesity care in Central Europe were included in this analysis (mean body mass index (BMI) 29.0 ± 5.6 kg m(-)(2), standard deviation score body mass index (SDS-BMI) 2.48 ± 0.54, 45% boys, age 11.4 ± 2.8 years). The changes of weight status, blood pressure, fasting lipids and glucose, and oral glucose tolerance tests were documented by standardized prospective quality documentation software (APV). RESULTS: After follow-up of in median 1.2 (interquartile range 0.9-2.2) years, a mean reduction of -0.15 SDS-BMI was achieved. The prevalence of prehypertension (37->33%) and hypertension (17->12%) decreased, while prevalences of triglycerides >150 mg dl(-1) (22->21%), low-density-lipoprotein-cholesterol >130 mg dl(-1) (15->14%), impaired fasting glucose (6->6%) and impaired glucose tolerance (9->8%) remained stable. Drug treatment according to cutoffs recommended in European obesity guidelines were not frequently indicated (hypertension: 10%; dyslipidemia: 1%, type 2 diabetes <1%). None of the children with dyslipidemia received lipid-lowering drugs and only 1.4% of the children with hypertension were treated with antihypertensive drugs. CONCLUSIONS: Achieving sufficient weight loss to improve obesity associated comorbidities was difficult in clinical practice. Drug treatment of hypertension, dyslipidemia and type 2 diabetes was rarely performed even if it was indicated only in a minority of the overweight children. Future analyses should identify reasons for this insufficient drug treatment of comorbidities and analyze whether the benchmarking processes of APV improve medical care of childhood obesity.

Monitoring of kratom or Krypton intake in urine using GC-MS in clinical and forensic toxicology.

The Thai medicinal plant Mitragyna speciosa (kratom) is misused as a herbal drug. Besides this, a new herbal blend has appeared on the drugs of abuse market, named Krypton, a mixture of O-demethyltramadol (ODT) and kratom. Therefore, urine drug screenings should include ODT and focus on the metabolites of the kratom alkaloids mitragynine (MG), paynantheine (PAY), speciogynine (SG), and speciociliatine (SC). The aim of this study was to develop a full-scan gas chromatography-mass spectrometry procedure for monitoring kratom or Krypton intake in urine after enzymatic cleavage of conjugates, solid-phase extraction, and trimethylsilylation. With use of reconstructed mass chromatography with the ions m/z 271, 286, 329, 344, 470, 526, 528, and 586, the presence of MG, 16-carboxy-MG, 9-O-demethyl-MG, and/or 9-O-demethyl-16-carboxy-MG could be indicated, and in case of Krypton, with m/z 58, 84, 116, 142, 303, 361, 393, and 451, the additional presence of ODT and its nor metabolite could be indicated. Compounds were identified by comparison with their respective reference spectra. Depending on the plant type, dose, administration route, and/or sampling time, further metabolites of MG, PAY, SG, and SC could be detected. The limits of detection (signal-to-noise ratio of 3) were 100 ng/ml for the parent alkaloids and 50 ng/ml for ODT. As mainly metabolites of the kratom alkaloids were detected in urine, the detectability of kratom was tested successfully using rat urine after administration of a common user's dose of MG. As the metabolism in humans was similar, this procedure should be suitable to prove an intake of kratom or Krypton.

[Treatment of extremely obese adolescents and young adults at the Insula Obesity Center in Bischofswiesen]. / Therapie der extremen juvenilen Adipositas im Adipositas-Zentrum Insula in Bischofswiesen.

The Insula Obesity Center has been treating extremely obese adolescents and young adults since 1992. Various programs ranging from 2-9 months' duration are offered. The mean BMI at admission has been increasing continuously and is presently 41.5 kg/m(2) with occasional extremes over 80 kg/m(2). Obesity comorbidities are common. A mean weight reduction of 1.3 kg/week is achieved during a mean duration of treatment of 4.7 months. Follow-up in residential support groups is offered for up to 2 years for selected patients with special challenges such as lack of family support.

Use of liquid chromatography coupled to low- and high-resolution linear ion trap mass spectrometry for studying the metabolism of paynantheine, an alkaloid of the herbal drug Kratom in rat and human urine.

The Thai medicinal plant Mitragyna speciosa (Kratom in Thai) is misused as a herbal drug of abuse. During studies on the main Kratom alkaloid mitragynine (MG) in rats and humans, several dehydro analogs could be detected in urine of Kratom users, which were not found in rat urine after administration of pure MG. Questions arose as to whether these compounds are formed from MG only by humans or whether they are metabolites formed from the second abundant Kratom alkaloid paynantheine (PAY), the dehydro analog of MG. Therefore, the aim of the presented study was to identify the phase I and II metabolites of PAY in rat urine after administration of the pure alkaloid. This was first isolated from Kratom leaves. Liquid chromatography-linear ion trap mass spectrometry provided detailed structure information of the metabolites in the MS(n) mode particularly with high resolution. Besides PAY, the following phase I metabolites could be identified: 9-O-demethyl PAY, 16-carboxy PAY, 9-O-demethyl-16-carboxy PAY, 17-O-demethyl PAY, 17-O-demethyl-16,17-dihydro PAY, 9,17-O-bisdemethyl PAY, 9,17-O-bisdemethyl-16,17-dihydro PAY, 17-carboxy-16,17-dihydro PAY, and 9-O-demethyl-17-carboxy-16,17-dihydro PAY. These metabolites indicated that PAY was metabolized via the same pathways as MG. Several metabolites were excreted as glucuronides or sulfates. The metabolism studies in rats showed that PAY and its metabolites corresponded to the MG-related dehydro compounds detected in urine of the Kratom users. In conclusion, PAY and its metabolites may be further markers for a Kratom abuse in addition of MG and its metabolites.

Adenovirus-mediated transfer of a recombinant alpha 1-antitrypsin gene to the lung epithelium in vivo.

The respiratory epithelium is a potential site for somatic gene therapy for the common hereditary disorders alpha 1-antitrypsin (alpha 1AT) deficiency and cystic fibrosis. A replication-deficient adenoviral vector (Ad-alpha 1AT) containing an adenovirus major late promoter and a recombinant human alpha 1AT gene was used to infect epithelial cells of the cotton rat respiratory tract in vitro and in vivo. Freshly isolated tracheobronchial epithelial cells infected with Ad-alpha 1AT contained human alpha 1AT messenger RNA transcripts and synthesized and secreted human alpha 1AT. After in vivo intratracheal administration of Ad-alpha 1AT to these rats, human alpha 1AT messenger RNA was observed in the respiratory epithelium, human alpha 1AT was synthesized and secreted by lung tissue, and human alpha 1AT was detected in the epithelial lining fluid for at least 1 week.

[Long-term inpatient treatment of extreme juvenile obesity: an 18-month catamnestic study]. / Studie zur stationären Langzeittherapie der extremen juvenilen Adipositas. Jeder Zweite nimmt langfristig ab.

Eighteen months after completion of long-term treatment of 98 extremely overweight juveniles in the rehabilitation center Insula, this study revealed an improvement in the age-specific body mass index (BMI-SDS) in 55.1% of the cases, when all the non-responders (approx. 22%) were evaluated as failures. An improvement of at least 0.2 or 0.5 BMI SDS points was achieved in 41.8% and 21.4%, respectively.

Independent confirmation of a major locus for obesity on chromosome 10.

Linkage results obtained in genome-wide scans for complex phenotypes require confirmation in independent samples. Recently, linkage of obesity to chromosome 10p12 with a maximal multipoint LOD score of 4.85 was reported upon use of an affected sib-pair approach including nuclear families in which the adult index case had a BMI > or = 40 kg/m2 and at least one further sibling had a BMI > or = 27 kg/m2 (Hager et al., 1998, Nat Genet 20:304-8). To attempt to replicate this linkage finding we genotyped 11 markers spanning approximately 23 cM from 10p13 to 10ql1 in a total of 386 individuals stemming from 93 nuclear families with two or more young obese offspring with a BMI > or = 90th age percentile. The highest multipoint maximum likelihood binomial (MLB) LOD score using the extreme concordant sib-pair approach in which one sib had a BMI > or = 95th percentile, and other sibs a BMI > or = 90th percentile was 2.32. Six markers yielded nominal p-values < 0.05, the highest two point MLB-LOD score of 2.45 (nominal p = 0.0004) was obtained for the marker TCF8. Transmission disequilibrium tests for the most frequent parental allele yielded no nominal p-value < 0.05. The linkage results confirm the presence of a major susceptibility locus for obesity in a region near the centromere on chromosome 10.

Systematic mutation screening of the pro-opiomelanocortin gene: identification of several genetic variants including three different insertions, one nonsense and two missense point mutations in probands of different weight extremes.

Pro-opiomelanocortin (POMC) is the precursor of melanocortins (adrenocorticotropin: ACTH, beta-endorphin, beta-lipotropin: beta-LPH, corticotropin like intermediate peptide, alpha-, beta- and gamma-melanocyte-stimulating hormone: alpha-, beta- and gamma-MSH) some of which act in the brain to reduce food intake and are potential mediators of leptin action. Recently, three different mutations in the POMC gene (POMC) were identified in two unrelated children that lead to early-onset extreme obesity, adrenal insufficiency, and red hair pigmentation. In the present study we systematically screened the coding region of POMC in 96 extremely obese children and adolescents, 60 healthy underweight individuals and 46 patients with anorexia nervosa (AN) and identified several variants. a) A 9 and an 18 base pair insertion (9bp and 18bp: AGC AGC GGC and AGC AGC GGC AGC AGC GGC, respectively, between codon 73 and 74; 1,2). These in-frame variants lead to the insertion of three or six amino acids (Ser-Ser-Gly; Ser-Ser-Gly-Ser-Ser-Gly) carboxy-terminal to gamma-MSH. Frequencies of the 9bp insertion allele varied between 3 and 5% among the different study groups (Pearson's chi2 P>0.5). b) Both an out-of-frame 6 bp insertion (within codon 176: GGG CCC) leading to the insertion of two amino acids (Arg-Ala) and a premature stop-codon (G-7316-T: Glu-180-Stop) within the gamma-LPH sequence were maternally inherited in an obese female proband. This proband inherited another missense mutation from her father (Glu-188-Gly). c) A missense mutation (G-7016-A; Asp-80-Asn) was observed in a single patient with AN who also harboured the 9bp insertion on a paternally derived haplotype. d) The allelic co-occurence of two silent mutations (C-6982-T and C-7285-T) was detected in two obese subjects. e) Two further silent mutations (C-3832-T; C-7111-G) were detected in an underweight and an obese subject, respectively. We conclude that the POMC gene harbors several different polymorphisms and mutations, none of which can readily be associated with the phenotypes under study.

Systematic mutation screening of the estrogen receptor beta gene in probands of different weight extremes: identification of several genetic variants.

Estrogens are known to have an inhibitory effect on food intake in rodents and primates. Decreased estrogen levels that are found for instance in menopausal woman and in ovarectomized rodents result in body weight gain. Estrogen can act both in the periphery and in the central nervous system via at least two different estrogen receptors (alpha and beta). We systematically screened the coding region and part of the 5' and 3'regions of the estrogen receptor beta gene (ER beta) in 96 extremely obese children and adolescents, 50 patients with anorexia nervosa (AN), 28 patients with bulimia nervosa (BN), and 25 healthy underweight individuals. We detected five different sequence variants in the ER beta: a) A 21 bp deletion (codons 238 to 244) was detected in two obese probands and an underweight individual. b) An 846G-->A transition leading to a nonconservative amino acid substitution (G-250-S) was found in two obese male probands. Both a) and b) were located within the flexible hinge region between DNA and ligand binding domain. c) For a 1082G-->A polymorphism we found suggestive evidence for an association between the more common 1082G-allele and anorexia nervosa (nominal p=0.04). d) One silent mutation (1421T-->C) was found solely in two obese probands. e) A common variant is located in the 3' nontranslated region at position 1730(A-->G). We did not detect association of this polymorphism to any of the analyzed phenotypes. We conclude that the ER beta harbors several different mutations and polymorphisms, none of which can readily be associated with the phenotypes under study.

Several mutations in the melanocortin-4 receptor gene including a nonsense and a frameshift mutation associated with dominantly inherited obesity in humans.

The melanocortin-4 receptor gene (MC4-R) has been implicated in weight regulation. Recently, two independent groups reported frameshift mutations associated with a dominant form of obesity (1, 2). We screened the coding region of the MC4-R in 306 extremely obese children and adolescents (mean body mass index: BMI 34.4 +/- 6.6 kg/m2), 25 healthy underweight students (mean BMI 17.1 +/- 0.8 kg/m2), 52 normal weight individuals (mean BMI 22.0 +/- 1.0 kg/m2), 51 inpatients with anorexia nervosa (AN, DSM IV criteria, mean BMI 14.3 +/- 1.5 kg/m2) and 27 patients with bulimia nervosa (BN, DSM IV criteria, mean BMI 21.7 +/- 5.8 kg/m2) by single strand conformation polymorphism analysis (SSCP). Several mutations were identified, including the frameshift mutation described (1). The mutations were as follows: a) The deletion of 4 bp (delta of CTCT at codon 211) results in a frameshift, thus rendering a truncated protein. This mutation has been assumed to be associated with dominantly-inherited morbid obesity in humans (1). Both the index patient (BMI 42.06 kg/m2, height 171 cm, age 19.6 years) and her mother (BMI 37.55 kg/m2, height 164 cm, age 42.5 years) were heterozygous for the deletion. b) A nonsense mutation at position 35 of the MC4-R was detected in two obese probands (BMI 31.29 kg/m2 and BMI 45.91 kg/m2). This mutation leads to a truncated protein that encompasses the N-terminal extracellular domain. Both carriers additionally showed (c) a missense mutation (Asp-37-Val). In both of these cases Tyr-35-Stop and Asp-37-Val were maternally transmitted, thus these variations form a haplotype. d) e) A male obese proband harbored two missense mutations (Ser-30-Phe, Gly-252-Ser). f)-i) Four different missense mutations (Pro-78-Leu, Thr-112-Met, Arg-165-Trp, Ile-317-Thr) were detected in four different male probands, respectively. All of these mutations (a to i) were found solely in extremely obese individuals whose BMIs were all above the 99th percentile. j) A silent mutation (C-579-T, Val-193-Val) was detected in a male underweight individual. k) A previously described polymorphism (Val-103-Ile; 3) was detected with similar frequencies in all different study groups. 1) We identified a novel polymorphism (Ile-251-Leu) with similar allele frequencies in all groups under study. In conclusion, our data indicate that mutations in the MC4-R are not uncommon. Whereas our data support the evidence for dominantly inherited obesity as revealed by the three obese probands with haplo-insufficiency, the functional significance of the missense mutations remains to be determined.

No evidence for involvement of polymorphisms of the dopamine D4 receptor gene in anorexia nervosa, underweight, and obesity.

Family and twin studies suggest a genetic contribution to the etiology of anorexia nervosa (AN) and obesity. Genes involved in weight regulation can be considered as candidate genes for AN. The dopaminergic system has been implicated in weight regulation; previous results had suggested a possible involvement of the dopamine D4 receptor gene (DRD4). We screened for alleles of two different polymorphisms (13-bp deletion, 48-bp repeat) in the DRD4. For association tests, allele frequencies were compared between 109 inpatients with AN, 82 underweight students, and 327 extremely obese children and adolescents. For application of transmission disequlibrium tests (TDT) we additionally genotyped 57 and 137 trios comprising a patient with AN or an extremely obese child or adolescent, respectively, and both parents. All genotyping was performed with polymerase chain reaction fragment length polymorphism analyses. None of the association tests or TDT rendered nominal P values below 0.1. An influence of alleles of the DRD4 on the development of AN, underweight, or extreme early onset obesity was not detected. Am. J. Med. Genet. (Neuropsychiatr. Genet.) 88:594-597, 1999.

Missense variants in the human peroxisome proliferator-activated receptor-gamma2 gene in lean and obese subjects.

The peroxisome proliferator-activated receptor-gamma2 (PPARgamma2) is almost uniquely expressed in adipose tissue and is of major importance for fat cell differentiation and lipid metabolism. This study was undertaken to assess whether two missense variants in the PPARgamma2 gene are associated with early-onset obesity. A previously described polymorphism encoding for an amino acid exchange in codon 12 (Pro12Ala) was detected with allele frequencies of 0.13 in 296 markedly obese children and adolescents and 0.14 in 130 lean individuals. A Pro115Gln variant, which had been linked to obesity in Germans in a previous association study, was not detected in any of our obese or lean subjects, who are also of German origin. We conclude from our data that these two variants in the PPARgamma2 gene are unlikely to contribute to the high prevalence of early-onset obesity.

Identification of a deletion variant in the gene encoding the human alpha(2A)-adrenergic receptor.

OBJECTIVE: The alpha(2)-adrenergic receptors are involved in the effects of catecholamines on energy metabolism. Of three known subtypes with differential expression, alpha(2A)-adrenergic receptors are also localized in adipose tissue where they counteract the lipolytic activity of beta-adrenergic receptors. This study was undertaken to assess whether variants in the alpha(2A)-adrenergic receptor gene are associated with body weight. DESIGN AND METHODS: Single strand conformation polymorphism (SSCP) screening and subsequent sequencing were applied to determine genetic variants in DNA samples from individuals with obesity, those of normal weight and those underweight. RESULTS: Analysis of the coding region resulted in the identification of an 18 bp deletion, with no other mutation found. Of 429 genotyped subjects, 7 carried the deletion, with no significant differences between lean and obese subjects. A previously identified polymorphism in the promoter of the alpha(2A)-adrenergic receptor gene also did not show an association with any of the tested body weight categories. CONCLUSION: Our data suggest that variants in the alpha(2A)-adrenergic receptor gene are unlikely to contribute to the predisposition for the lean or obese state.

Phosphatidylinositol transfer protein in murine embryonal carcinoma cells during retinoic acid-induced differentiation.

Phosphatidylinositol transfer protein (PI-TP) was studied in P19 embryonal carcinoma (EC) cells at different stages of retinoic acid (RA) induced differentiation. Western blot analysis indicated an increased expression of PI-TP (35 kDa) during differentiation. Western blots of isoelectric focusing gels showed that the 35 kDa band consisted of the PI-carrying form of PI-TP (pl 5.5) and of a novel, more acidic form of PI-TP (pl 5.4), levels of both of which increased during differentiation. These increased levels were not reflected in the in vitro PI-transfer activity of the cytosolic fraction nor in the mRNA levels as analyzed by northern blotting. By using indirect immunofluorescence it was shown that PI-TP is localized in the cytoplasm and associated with perinuclear Golgi structures and that this distribution is slightly affected during RA-induced differentiation. Immunoprecipitation of PI-TP from [32 P]Pi labeled cells demonstrated that the level of phosphorylation of PI-TP is high in undifferentiated P19 EC cells and low after 5 days of RA-induced differentiation. These results strongly suggest that changes in the levels of PI-TP are intimately connected with changes in the growth characteristics of P19 EC cells during RA-induced differentiation. It remains to be established to what extent this connection is governed by the recent finding that PI-TP is an essential cytosolic factor in stimulating phospholipase C activity.

Levels of 5'-nucleotide phosphodiesterase isoenzymes in normal and pathological sera.

The levels of 5'-nucleotide phosphodiesterase isoenzymes (5'-NPD; EC 3.1.4.1) in sera of 54 healthy donors and 201 inpatients were measured. Isozymes were separated electrophoretically and designated as 5'-NPD-0, -I, -II, -III, -IV and -V in the reverse order of their electrophoretic mobility. In healthy donors all isozymes except 5'-NPD-V were detectable. In pathological sera isozymes 0 to V were elevated in 26.0%, 20.5%, 14.0%, 30.5%, 7.0% and 15.0% of the cases, respectively. Decreased values were found in 6-7%, with the exception of 5'-NPD-IV showing decreased activities in 23.5% of the patients. This average distribution pattern was found in many disorders. However, in diseases of the liver and the pancreas a remarkable accumulation of cases with elevated levels of all isozymes, except 5'-NPD-IV, was observed. All isozymes, except 5'-NPD-IV, showed many significant correlations with other laboratory parameters indicating liver disease. Isozyme IV was not related to these parameters but exhibited a strong correlation with serum albumin. 5'-NPD-II was unproportionally often increased in patients with liver cirrhosis and was the only isozyme with on the average higher levels in women than in men.

No evidence for involvement of the promoter polymorphism -866 G/A of the UCP2 gene in childhood-onset obesity in humans.

Recently, an association between obesity and the G-allele of the - 866 G/A polymorphism in the promoter region of uncoupling protein-2 gene (UCP2) was reported. Both allele frequencies and genotype distributions for this polymorphism differed between obese individuals and never-obese controls. We attempted to confirm this finding. Genotyping was performed by polymerase chain reaction with subsequent restriction fragment length polymorphism analysis (PCR-RFLP). We analysed transmission disequilibrium of the (wild type) G-allele for 200 extremely obese children and adolescents from 93 concordant sib pair families using the pedigree disequilibrium test. Additionally, using a one-sided asymptotic Pearson's chi 2-test, we tested whether the G-allele occurs more frequently in 277 extremely obese children and adolescents (including the 93 index patients of the concordant sib pairs) than in 188 never-obese controls. The one-sided asymptotic Cochran Armitage trend test was used to determine differences in genotype frequencies between extremely obese and healthy underweight individuals. The PDT analysis revealed no evidence for transmission disequilibrium in obesity. Allele and genotype frequencies did not differ between the extremely obese and never-obese subjects. In conclusion, we cannot confirm the results of ) in our young sample.